Homogeneous vaccine composition for the tumor treatment and its obtaining method

ABSTRACT

The present invention relates to the biotechnological field and particularly to the human health. More particularly, the present invention relates to a vaccine composition for therapeutic use in cancer patients. 
     The vaccine composition of the present invention has as active principle a chemical conjugated between the human recombinant Epidermal Growth Factor (hrEGF) and the P64K recombinant protein. In another embodiment, the present invention relates to the conjugation procedure to obtain, a chemical conjugated under controlled and reproducible parameters. 
     In a preferred embodiment, the present invention relates to the procedure for purifying the chemical conjugated with a higher purity of the therapeutical vaccine composition, and a surprisingly increased immunogenic activity, inducing a significant increase of the anti-EGF antibody titers in humans. 
     Additionally the present invention provides the methodology to obtain a vaccine composition in several dose presentations (total milligrams of conjugated EGF-P64K/vial). The versatility of the dose presentation allows increasing the immunization dose per patient, without increasing the frequency of injections and/or immunization sites. 
     Besides, the present invention relates to a sanitary procedure for obtaining a vaccine composition for parenteral route in the cancer therapy.

TECHNICAL FIELD

The present invention relates to biotechnology, particularly with thehuman health. In an embodiment, the present invention describesprotective and for therapeutic cancer vaccines, principally vaccinecompositions to elicit a significant increase of the immune responseagainst the Epidermal Growth Factor (EGF), whose oncological relevancehas been broadly demonstrated, mainly for the growth of tumors ofepithelial origin.

PREVIOUS ART

The EGF receptor (EGFR) system, including the ligands, is a molecularcomplex which specifically regulates the cellular growth and its effecton the uncontrolled growth of epithelial tumors.

In the tumorigenic process, the deregulation of paracrine and autocrineprocesses for activation of the EGRF, involve the over-expression of thegrowth factors as well as the increased synthesis and/or mutation of thereceptors.

The EGF is a 53 amino acids polypeptide, with an apparent molecularweight of 6045 Da. This peptide was isolated and purified for the firsttime by Cohen S., J. Biol. Chem. (1962) 237, 1.555).

The EGF is a member of the EGFR ligands family; this family comprisesstructural and functionally related proteins. Others members of thisfamily are: Transforming Growth Factor (TGF), anmphiregulin (AR),criptol (CR1), heparine binding growth factor, betaceluline andephireguline. On the other hand the family of the poxvirus includesproteins related with the EGF; among them the most characterized is thevaccinia virus growth factor (VGF).

All these molecules are able to bind the EGFR with the consequentreceptor activation, so they are known as EGFR ligands and play a rolein the normal and tumor cell growth.

The EGFR is a 170 kDa glycoprotein; the gene has already been cloned andsequenced. The intra-cellular domain of this receptor is associated withthe tyrosine kynase activity of molecules that shows a structuralhomology with those proteins coded by the v-erb-B oncogen, and they areinvolved with the malignant transformation process (HELDIN C. H. (1984),CELL 37, 9-20.).

The experimental evidences of the last years about the relation betweenthe EGFR and its ligands system with cancer, makes it a very attractivetarget for cancer immunotherapy.

Previous results have demonstrated the efficacy for cancer of the activeimmunotherapy with the EGF based vaccine. In fact, preclinical andclinical evidences have been obtained about the immunogenicity and lackof toxicity caused by vaccination with the hrEGF linked to a carrierprotein (González and cols. (1996), Vaccine Research 5(4), 233-243.)

EP 0 657 175 describe a vaccine composition that comprises autologousEGF coupled to carrier protein which inhibit the growth of EGF dependingtumors by an auto-immune effect.

The vaccine composition described in EP 0 657 175, is conjugatedvaccines comprising the EGF coupled to a carrier protein (the P64Kprotein, the cholera toxin B chain, the titanic toxoid protein and/ormonoclonal antibodies) and as a consequence is obtained an heterogeneousand low reproducible mixture of conjugated species.

The novel vaccine composition described in the present inventioncomprises the autologous EGF as active principle, and is characterizedfor having a homogeneous chemical composition, with a defined purity,eliciting higher immunogenicity, a substantially increased clinicalactivity and with fewer immunizations for getting a therapeuticaleffect.

The lower autologous antigen content in the vaccine composition of thepresent invention surprisingly doesn't induce lower EGF antibody titersin humans by the contrary, it rather induce significant increase of theanti-EGF antibody titers. The lower amount of EGF contents and itsderivatives in the vaccine composition was obtained by developing of apurification method based on membrane filtration, which is also anobject of the present invention.

Furthermore, the present invention provides a homogeneous andreproducible vaccine composition with a higher clinical effect and lowerimmunizations, meaning a great advantage in cancer therapy and for thepatient, in comparison with vaccine compositions previously described.

The vaccine composition of the present invention could also compriseappropriate adjuvants such as Aluminium hydroxide or Montanide.

In another embodiment, the present invention relates to a sanitaryprocedure for the manufacture of said vaccine composition, appropriatefor its use in humans by parenteral route.

The procedure comprises a suitable method for the covalent conjugationof the hrEGF to the carrier protein P64K, and the purification of thisconjugated by using ultra filtration membranes in a range of 50-100 kDa,to remove the biologically inactive species conjugated (withoutimmunogenic activity), as well as other chemical impurities.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a novel vaccine composition based on theautologous EGF having said vaccine composition a homogeneous chemicalcomposition with defined purity, and which is able to elicit potentateimmunogenicity and a substantially increased clinical activity.Additionally, with the vaccine composition of the present invention areable higher therapeutic doses without increasing the number ofinjections when the therapeutic dose must be increased, because by theprocedure to obtain the vaccine composition of the present invention itis possible to increase the concentration of the conjugated specieshrEGF-rP64K, which is known as potency.

Surprisingly, the inventors found that when decreasing the content ofthe autologous antigen by a novel purification procedure the vaccinecomposition of the present invention is able to elicit a significantincrease of the immune response against the autologous EGF in comparisonwith heterogeneous EGF-based vaccines previously reported.

The vaccine composition obtained from the decrease of the content of theautologous antigen through the membrane ultrafiltratrion purificationmethodology has a homogeneous composition where the immunologicallyactive species (conjugated species hrEGF-rP64K) shows molecular weightsuperior to 60 kDa. This vaccine composition shows a molecular exclusionchromatographic profile characterized because the species carrying thebiological activity represents more than the 90% of the totalchromatogram area, corresponding to the total vaccine composition, as itis shown in FIG. 4

Mass Spectrometry analysis of the present vaccine composition hasrevealed its structural features. The peptide maps of two different lotsshow a high homology between lots in the position s as well as in theratio (FIG. 5). This high homology between lots confirms the accuracy ofthe parameters of the chemical conjugation as well as the purificationprocedures, both described by the present invention. Mass Spectrometryanalysis further revealed the sequence of the major peptides derivedfrom the vaccine composition of the present invention. (Table 3)

In the present vaccine composition the conjugation ratio between the EGFand P64K proteins is 1:2, which means 2 molecules of EGF per P64Kmolecule.

Methodology for Obtaining the Vaccine Composition. PurificationProcedure for Decreasing the Content of Autologous EGF.

Starting from experimental observations that evidenced that a decreaseon the hrEGF content in the vaccine composition increase itsimmunological action, is developed a method for decrease the content inhrEGF in the heterogeneous mixture of conjugates; enriching the vaccinecomposition in conjugated species of high molecular weight(immunologically active species) and free of glutaraldehyde.

The purification procedure using ultrafiltration membranes in a rangeamong 50-100 kDa developed and described in the present invention,consists of two stages:

In the initial stage successive changes of tampon solution are performed(diafiltration) to remove glutaraldehyde and to eliminate the excess ofautologous protein, either free or forming conjugated hrEGF-hrEGF ofdifferent sizes. During this stage, between 10 and 15 changes of thetampon solution are carried out.

The second stage is the concentration of the purified chemicalconjugated, in which more than 90% of its composition corresponds toimmunogenic species hrEGF-rP64K. During the concentration stage theinitial volume of the chemical conjugated is reduced until reaching afinal protein concentration (conjugated species hrEGF-rP64K) in a rangeamong 1-12 mg/mL. This versatility in the range of concentrations of theactive principle (total milligrams of conjugated hrEGFrP64K formilliliters) for this vaccine composition is a great advantage forcancer therapy. This allows increasing the treatment dose withoutimplying for the patient an increment in the frequency of immunizationand/or an increase in the number of immunization sites.

With the purpose of monitoring the quality of the purification of thechemical conjugated it was evaluated the pH and the conductivity of theone retained and of the permeate. It was also carried out thedetermination of protein concentration by the method described by LowryD. H. and col. J. Biol Chem 191: 495-498, 1951 and the percent ofhomogeneity were determined from the analysis of the gel filtrationchromatography (HPLC-FG).

The singularity of this methodology assures that the obtained vaccinecomposition has a homogeneous composition and defined purity,characterized by the major presence of the immunologically activespecies: conjugated hrEGF-rP64K.

Surprisingly, the removal of the excess of autologous protein (hrEGF)free or polymeric doesn't cause the reduction, but rather it allows toincrease significantly the concentration of anti-EGF antibodies (at1/1000 dilution), to 10 times more this obtained when immunizing micewith polymers of hrEGF and with free hrEGF. The reduction of the contentof autologous protein in the vaccine composition causes in patientincrements of at least 2 times the maximum titer of anti EGF antibodies.This increment of the antibody titers is even observed in those patientsthat receive half of per immunization (2.4 mg) in comparison withpatients that receive dose of 4.8 mg per dose of the vaccine compositiondescribed in EP 0657 175. These results indicate that the vaccinecomposition, described in the present invention has bigger immunologicalactivity per milligram of immunologically active protein, as comparedwith other vaccine compositions.

Obtaining a Chemical Conjugated Between the Recombinant Protein P64K(rP64K) and the Human Recombinant Epidermal Growth Factor (hrEGF).

Starting from the evaluation and optimization of the conditions for thechemical conjugation reaction between the proteins hrEGF and rP64K aconjugation method is developed that requires of a high molar proportionof the autologous protein, 10 moles of hrEGF for each rP64K mol, toguarantee high conjugation efficiency among both. The necessary excessof autologous protein during the chemical conjugation, to guarantee itsefficiency, is eliminated subsequently through the purification methodof membrane ultrafiltration as previously described, that allowsobtaining a vaccine composition with high homogeneity. The proceduredescribed in the present invention to guarantee an appropriate chemicalconjugation among both proteins consists of a single step and beginsmixing the hrEGF protein previously concentratiod (>6 mg/mL) and theprotein rP64K (≧1 mg/mL) in the conjugation reactor. To this proteinmixture is added the PBS/MgCl₂ solution (pH 6.8-7.2) and the conjugationsolution of glutaraldehyde 0.5%. The mixture is maintained in constantstirring for 2 hours at temperature 22° C.±2° C. The final proteinconcentration in the reaction mixture for the hrEGF is 0.82 mg/mL andfor the protein rP64K it is 0.89 mg/mL. The total protein concentrationduring the conjugation reaction is of 2 mg/mL and the finalconcentration of the glutaraldehyde in the reaction mixture is of 0.05%.

The vaccine composition according to the present invention can beemployed together with appropriated adjuvants, such as aluminumhydroxide or Montanide.

In another aspect, the present invention involves a sanitary procedurefor obtaining of a vaccine composition, to be administered by parenteralroute, procedure that minimizes the opportunities of microbialcontamination of the vaccine composition during the conjugation allowingto carry out the purification methodology of the chemical conjugated infew hours and facilitating the increment of the volume of the vaccinecomposition to be obtained, among other advantages. The followingexamples illustrate more in detail the present invention.

The Example 1 describes the molecular characterization of the vaccinecomposition described in the present invention. The molecularcharacterization includes: the chromatographic identification by HPLC-FGof the conjugated species formed during the conjugation reaction, thedetermination of the conjugation ratio between the proteins hrEGF andrP64K and the definition of the peptides map that characterizes to thisnovel vaccine composition.

The Example 2 describes the bio-assays in mouse carried out to evaluatethe immunogenicity of the novel vaccine composition described in thepresent invention.

The Example 3 describes the effectiveness in the clinical use of thepresent vaccine composition in the treatment of lung tumors ofepithelial origin.

The Example 4 describe obtaining the vaccine compositions adjusted a atdifferent potency (total milligrams of conjugated hrEGF-rP64K/vial),using the same conjugation methodology and purification as described inthe present invention.

The Example 5 shows the removal of the glutaraldehyde used in theconjugation reaction by means of the purification methodology of thechemical conjugated through a 50 kDa ultrafiltration membrane.

EXAMPLES Example 1 Molecular Characterization of the Vaccine CompositionDescribed in the Present Invention (Vaccine Composition A) 1.1Chromatographic Characterization of the Vaccine Composition Described inthe Present Invention.

For the chromatographic characterization of the vaccine compositionpurified by ultra filtration, homoligomeric conjugated hrEGF-hrEGF andrP64K-rP64K were used as indicators

The chromatographic profiles shown by the homoligomeric conjugated(hrEGF-hrEGF and rP64K-rP64K) were compared with the chromatographicprofile shown by the conjugated without purifying obtained by thechemical conjugation reaction among the proteins hrEGF and rP64K. InFIG. 1 are shown the profiles of the homoligomeric conjugatesrP64K-rP64K and the profile obtained when carrying out the chemicalconjugation between the proteins hrEGF and rP64K (chemical conjugatedwithout purifying by ultrafiltración). The circulated area belongs tothe conjugated species hrEGF-rP64K. The profile of the conjugatedrP64K-rP64K doesn't show total coincidence with this of the chemicalconjugated hrEGF-rP64K due to the contribution of the hrEGF moleculesconjugated to the rP64K.

In FIG. 2 are shown the results obtained after carrying out anun-naturalized electrophoresis in reduced conditions and a Western Blotof the vaccine composition purified by ultrafiltración. The developmentwas carried out with an anti-EGF antibody conjugated with alkalinephosphatase. The EGF is a protein of apparent molecular weight of 6 kDaso; it should be located in the inferior area of the gel. Itidentification by means of immuno-detection with anti-EGF antibodies inthe area corresponding to the proteins of molecular weigh apparentlysuperiors to 6 kDa confirms that hrEGF molecules exist together to therP64K in the area of the gel corresponding to the conjugated specieshrEGF-rP64K.

When comparing the chromatographic profile of the chemical conjugatedhrEGF-rP64 without purification with this of the homoligomericconjugated hrEGF-hrEGF, it can be appreciated that exist acorrespondence between both profiles. This evidence confirm that afterthe valley, exist a zone that corresponds to the hrEGF speciesconjugated in between them (EGF polymers) as well as free hrEGF (FIG.3).

The molecular exclusion chromatographic profile (FIG. 4) of the vaccinecomposition object of the present invention shows that the areacorresponding to the conjugated species hrEGF-rP64K, constitute morethan the 90% of the total area of the chromatogram (shadow area)evidencing the high homogeneity of the described vaccine composition.

1.2. Conjugation Ratio Between the Proteins hrEGF and rP64K in theVaccine Composition Described in the Present Invention.

With the objective of determining the conjugation ratio of the hrEGF andthe rP64K in the vaccine composition, the moles of each one of theconstitutive proteins were determined For this it was taken a vaccinecomposition obtained from the 50 kDa ultrafiltration process, and thechromatographic fraction corresponding to the rhEGF-rP64K conjugatedspecies was collected. These fractions were subjected to amino acidanalysis.

The determination of the conjugation ratio was carried out by 2 methods.

-   -   1. A method was based in the estimation of the quantity of        Phenylalanine (Phe) and Threonine (Thr) in the vaccine        composition (aminoacid only present in the rP64K molecule). The        quantities of these amino acids were used to determine the        quantity of rP64K. Starting from this last one, and keeping in        mind the total quantity of amino acids of the mixture, the        quantity of EGF was determined.    -   2. The other method was based on the direct determination of the        relative quantities of each protein, using the amino acid        sequence and based in the quantification of those amino acids        Asparagine+Aspartic (Asx), Glutamine+Glutamic (Glx), Glicine        (Gly) and Alanine (Ala) (residues highly stable to the acid        hydrolysis and commonly used in the quantificatión of proteins).

Tables 1 and 2 show the results obtained by each method.

TABLE 1 Determination of the conjugation ratio between the hrEGF andrP64K proteins carrying out the determination of aminoacidic compositionof the vaccine composition purified by ultra- filtración, based on themethod of determination of the quantity of Phenilalanina (Phe) andThreonina (Thr) Mean ratio of Mean quantity of rP64K Mean quantity ofhrEGF conjugation 12.07 nmoles 23.13 nmoles 1.92

TABLE 2 Determination of the conjugation ratio between the hrEGF andrP64K proteins carrying out the determination of aminoacidic compositionof the vaccine composition purified by ultrafiltration, based on themethod of determination of the relative quantities of the amino acidsAsparagine + Aspartic (Asx), Glutamine + Glutamic (Glx), Glicine (Gly)and Alanine (Ala) Protein Quantity of protein Conjugation ratio rP64K12.04 nmoles 2.00 hrEGF 24.05 nmoles

The results obtained by both methods shows correspondence in theestimate of the conjugation ratio among the hrEGF:rP64K proteins for thevaccine composition described in the present invention. The calculatedratio of conjugation was of (1:2), which means 2 hrEGF molecules foreach rP64K molecule.

1.3. The Peptide Maps Characterizing the Vaccine Composition Describedin the Present Invention.

With the objective of characterizing the structure of the vaccinecomposition presented in the present invention and to evidence thereproducibility of the procedure it was developed and characterized thepeptide map obtained after the digestion of the conjugated fractionshrEGF-rP64K with the endoprotease Glu-C. Each one of the fractions ofthe obtained map were identified and sequenced by Mass Spectrometry.FIG. 5 shows the peptide maps obtained starting from two vaccinecompositions obtained independently, where a great similarity isappreciated in the peptides that appeared and in its relativeproportion. The reproducibility among those peptide maps obtainedstarting from both vaccine compositions is an indicative of the controlthat exist over the conditions in which the procedures of chemicalconjugation and purification (described in the present invention)happen.

The identification of the peptides contained in each fraction of the mapwas obtained by the analysis of each one of them by the MALDI method(Matrix Assisted Laser Desorption Ionization) and by the ESI-MS method(Electrospray Ionization/Electronebulization by ionization). In Table 3the sequence of the major peptides is shown.

TABLE 3 Aminoacidic composition of the major peptides obtained after thedigestion of the EGF-P64K conjugated fraction with the endoprotease Glu-C. The aminoacidic composition was determined by MALDI and ESI-MS. PeakPeptides HG05 LDID QAAPTGE HG06 SIGMAAE AEGTAAAPKAE HG08 AEGTAAAPKAEHG09 KISE SIGMAAE HG11 AAAAPAQEAPKAA GANAPKEPQRYD HG12 SIGMAAE HG14VKVKVGDKISE HG15 VAWVGE HG16 VAWVGETE VSLTAGDAYE HG17 VCLAIE HG18VRHLAANGIKYPEPE HG19 AAAAPAQEAPKAAAPAPQAAQFG VRHLAANGIKYPEPE HG20LKVPDIGGHE RVIPGVAYTSPE TGRIIGGGIVGPN VRHLAANGIKYPEPE HG21 KAGVAVTDRGFIELIFDAE GGPGGYSAAFAAADE HG22 ARVIPGVAYTSPE GLKVAIVE HG23AAAAPAQEAPKAAAPAPQAAQFGGSADAEY D MGCDAADIGKTIHPHPTLGE PKEPQRYDAVLVAAGRHG24 NVDIIAVE YDVVVLG HG25 AVLVAAGRAPNGKLISAE MGCDAADIGKTIHPHPTLGETGRIIGGGIVGPNGGDMIGE HG26 TGRIIGGGIVGPNGGDMIGE VRHLAANGIKYPEPELD HG27TGRIIGGGIVGPNGGDMIGE RCQYRDLKWWE HG28 GGLIVVVE HG29 GGLIVVVETGRIIGGGIVGPNGGDMIGE YRFDNIMVNTKTVAVEPKED HG30 AIGDIVGQPMLAHKAVHEAIGDIVGQPMLAHKAVHE HG31 ALDKYACNCVVGYIGE ALDKYACNCVVGYIGE HG32ARVIPGVAYTSPE LDIDMLRAY MDVPAEVAGVVKE HG33 TGRIIGGGIVGPNGGDMIGEALDKYACNCVVGYIGE GYCLHDGVCMYIE RCQYRDLKWWE HG34 NCAGHKAYFDARVIPGVAYTSPEHG35 GANAPKEPQRYDAVLVAAGRAPNGKLISAE HG36 GANAPKEPQRYDAVLVAAGRAPNGKLISAENCAGHKAYFDARVIPGVAYTSPE VIDEVRHLAANGIKYPE HG37 MGTVYSTLGSRLDVVE HG38IIGGGIIGLE MDVPAEVAGVVKEVK NSDSECPLSHDGYCLHDGVCMYIE HG39VVVLGGGPGGYSAAFAAADE HG40 MGTVYSTLGSRLDVVE HG41 LKVPDIGGHENVDIIAVE HG42RCQYRDLKWWEL HG43 VDKQMRTNVPHIYAIGDIVGQPMLAHKAVHE ASGRAIANGCDNGFTKLIFDAEHG44 VDKQMRTNVPHIYAIGDIVGQPMLAHKAVHE HG47 YRFDNIMVNTKTVAVEPKEDGVYVTFEAIANGCDNGFTKLIFDAE HG48 RYKTLGGVCLNVGCIPSKALLHNAAVIDE HG49VNVGDTIAVDDTLITLD RYKTLGGVCLNVGCIPSKALLHNAAVIDEYRFDNIMVNTKTVAVEPKEDGVYVTFE IVGQPMLAHKAVHEGHVAAENCAGHKAYFDNGFTKLIFDAETGRIIGGGIVGPNGGDMIGE HG50 TGRIIGGGIVGPNGGDMIGEVCLAIEMGCD HG51RCQYRDLKWWEL

Example 2 Immunogenicity of the Vaccine Composition Described in thePresent Invention

In this example is described the biological activity assay carried outfor evaluation of the immunogenicity of the vaccine compositiondescribed previously (vaccine composition A) in comparison with avaccine composition described in EP 0657 175 (called vaccine compositionB)

For the assay it was carried out the inoculation of 10 mice with anunique dose of 200 μL of a water in oil emulsion (50/50% v/v) of theimmunogens (conventional vaccine composition obtained by dialysis,vaccine composition obtained by purification with ultrafiltrationmembrane and permeated obtained from purification process by UF/DF whereconjugated species hrEGF-hrEGF are present), with the adjuvant MONTANIDEISA 51. The protein dose applied for each immunogen is referred in Table4.

In the 14 days following the inoculation it was carried out the serumextraction from the animals and the evaluation of the anti-EGF antibodytiters by means of an ELISA. The positive criterion for the assay wasthat the measured optical density at 405 nm must be higher than thedouble of the mean value obtained for the blank. The blank is obtainedwhen adding to the well blockade buffer instead serum. The test ofMann-Whitney was applied to the analysis of the data, by using astatistical program.

TABLE 4 Immunogens evaluated and protein concentrations to be inoculatedby animal according to the corresponding for the of biological activityassay. Protein concentration Immunogens (Lowry) Vaccine composition A1.5 mg/mL Vaccine composition B 1.5 mg/mL Homopolymeric conjugates ofhrEGF and 0.6 mg/mL hrEGF free obtained from the purification by UF/DFof the vaccine composition A

In FIG. 6, the results of the biological activity assay are shown asevidences of the immunogenic activity of each evaluated immunogen. Thecomponents of the immunogen corresponding to the homopolymeric hrEGFconjugates and free hrEGF, doesn't show relevant biological activity,because their response is at the level of the positive criteria for theassay or inferior. These results verify the hypothesis that the EGF byitself doesn't induce an immunogenic response.

The statistical analysis of all the results when applying theMann-Whitney Test'(Table 5) showed highly significant differences forthe anti-hrEGF antibody titers 1/1000 with p=0.0004 and for the 1/100titer with p=0.0006 for group of animals inoculated with the vaccinecomposition A as compared to the titers reached for the group of animalsinoculated with the vaccine composition B. This indicates that thevaccine composition A, purified by means of an ultrafiltration membranehas bigger immunogenic activity per milligram of protein that thevaccine composition B, obtained by the purification method based ondialysis membrane.

The vaccine composition A produces an increment of the concentration ofantibodies anti EGF (at 1/1000 dilution) 10 times higher as comparedwith this reached when immunizing with the conjugates corresponding topolymeric hrEGF and free hrEGF.

When comparing with the vaccine composition previously described in EP0657175 the vaccine composition described in the present inventionproduced an increment of twice the concentration of anti-rhEGFantibodies.

TABLE 5 Statistical report: the Mann-Whitney test used to evaluate theimmunogenicity of both vaccine compositions through the anti-hrEGFantibody titers in mice. Statistical analysis about the anti-hrEGFantibody titers in mice. (dilution 1/1000) Mann-Whitney Test and CI: G11/1000; G3 1/1000 N Median G1 (Preparado Vacunal B) 1/1000 20 0.4300 G3(Preparado vacunal A) 1/1000 20 0.7120 Point estimate for ETA1-ETA2 is−0.3115 95.0 Percent CI for ETA1-ETA2 is (−0.4500; −0.1790) W = 278.0Test of ETA1 = ETA2 vs ETA1 not = ETA2 is significant at 0.0004 The testis significant at 0.0004 (adjusted for ties) Statistical analysis aboutthe anti-hrEGF antibody titers in mice. (dilution 1/100) Mann-WhitneyTest and CI: G1 1/100; G3 1/100 N Median G1 (Preparado vacunalconvencional) 1/100 20 1.0785 G3 (Preparado vacunal por ultrafiltración)1/100 20 1.3040 Point estimate for ETA1-ETA2 is −0.2590 95.0 Percent CIfor ETA1-ETA2 is (−0.4389; −0.1149) W = 282.5 Test of ETA1 = ETA2 vsETA1 not = ETA2 is significant at 0.0006 The test is significant at0.0006 (adjusted for ties)

Example 3 Evaluation of the Clinical Efficacy of the Vaccine CompositionDisclosed by the Present Invention in the Treatment of Lung Tumors ofEpithelial Origin

The objective of this example is to evaluate in patients theimmunogenicity elicited by the Vaccine composition A in comparison withthe immunogenicity induced by the Vaccine composition B obtained by themethod of purification by dyalisis.

In this study patient with advanced stages Non Small Cell Lung Cancer(NSCLC) tumors were treated. Two groups of patients were defined(patients vaccinated with the preparation B and patients vaccinated withthe preparation A described in the present invention). The vaccinecomposition dose per patient in each group was as follows: Patientsvaccinated with the preparation B received 1.2 mg of total protein perimmunization site, while patients vaccinated with the preparation Breceived 0.6 mg of total proteins per immunization site. For both groupsthe immunization sites were 4. All patients received oncospecifictherapy, at least 4 weeks before starting the trial. Immunizations wereperformed by intramuscular route and continued occurring monthly.

The humoral immune response of both groups of patients was evaluatedthrough their serum. The main variable for evaluating the results wasthe specific anti-EGF antibody titer. This parameter was determinedthrough an ELISA. The positive values were those that gave opticaldensity readings 2 times higher than the negative control. The reportedantibody titer is the maximal dilution of positive serum.

The analysis of the immune response for patients in each group, asrepresented in Table 6, showed that the geometric mean of the maximalantibody titer in the group of patients treated with the preparation Acorresponds to 1:56421, while in the group of patients immunized withthe preparation B was of 1:23515. That means that the vaccinecomposition described in the present invention elicited an increase oftwice the geometric mean of the maximal antibody titers as compared withthe titters reached with the preparation B.

Historically the not immunized patients show an anti-autologous EGFantibody titer of 1:500. Therefore the vaccine composition described inthe present invention (vaccine composition A) causes an increase of atleast 100 times the title of anti-EGF antibodies in non-immunizedpatients.

TABLE 6 Result of the analysis of the geometric mean of immunogenicityfor both vaccine compositions through the measurement of anti-EGFantibody titers in patient's serum. Total of patients Geometric meanVaccine composition A 11 56421 Vaccine composition B 9 23515

It is important to stress that the group of patients immunized with theVaccine composition A, described in the present invention, showed asuperior geometric mean of the anti-EGF antibody titers receiving halfof the quantity of total proteins per immunization (2.4 mg) that the onereceived by the group of patients immunized with the Vaccine compositionB (4.8 mg of total proteins), what constitutes a surprising result.These results evidence that the excess of autologous protein hrEGF,either in form of polymers or free, doesn't contribute to moreimmunogenicity of the vaccine composition, but it dilutes theimmunological action of the chemical conjugated hrEGF-rP64K.

Example 4 Obtaining the Vaccine Composition Adjusted to DifferentPotency Concentration of Total Proteins Per Vial

With the purpose of demonstrating the capacity to obtain vaccinecompositions with different potency (concentrations of total proteinsper vial) it was carried out the purification methodology like it isdescribed below:

Assay 1: When concluding the diafiltración the purified chemicalconjugated was concentrated until a final concentration of proteins of 2mg/mL.

Assay 2: When concluding the diafiltración the purified chemicalconjugated was concentrated until a final concentration of proteins of 5mg/mL.

Assay 3: When concluding the diafiltración the purified chemicalconjugated was concentrated until a final concentration of proteins of12 mg/mL.

In all the assays the total volume of chemical conjugates employee was1500 mL, it was operated at a trans-membrane pressure of 1.5 bar and 7tampon solution changes were carried out during the diafiltracion. Forthe realization of this evaluation two selection criteria wereestablished:

The first one was to reach a homogeneity percent of the vaccinecomposition >80%. The second criterion was the non-presence ofprecipitated proteins during the purification operation.

The results, presented in Table 7, shows the vaccine compositionsadjusted at different protein concentrations per mL, guaranteeing highlevels of homogeneity (90.92%, 96.79% and, 95.50% of species conjugatedhrEGFrP64K) and without producing precipitation of proteins.

TABLE 7 Values corresponding to the percent of homogeneity in thevaccine compositions adjusted to different final protein concentrations.Percent of homogeneity of the Protein concentration vaccine composition2 mg/mL 92 00% 5 mg/mL 96.79% 12 mg/mL  95.50%

Example 5 Glutaraldehyde Removal from the Vaccine Composition of thePresent Invention

To evaluate the removal of the conjugation agent (glutaraldehyde) it wasquantified the residual glutaraldehyde contents in the vaccinecomposition B and in the vaccine composition A, by means of a reversephase chromatographic separation method (HPLC-RP), employing a C-8column.

This method required firstly, the precipitation of the protein containedin the samples with perchloric acid and later a reaction withphenylhydracine.

FIG. 7 show that the residual content of this impurity in both vaccinecompositions was smaller than 0.2 ug/mL or smaller than 0.002 ppm,(highest concentration limit established for this impurity) from aninitial concentration of 530 mg/mL or 530 ppm. Even when bothpreparations fulfill the established specification for this impurity,FIG. 7 illustration that the vaccine composition A (obtained whenapplying the ultrafiltration method for purification of the chemicalconjugated) possesses an inferior content of glutaraldehyde, whatcontributes to a great security for the patient that uses this vaccinecomposition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Shows the overlapping of the chromatogram of a homoligomericconjugated rP64K-rP64K with the chromatogram of a not purified chemicalconjugated EGF-P64K. In the circle is shown the coincidence of profilesbetween both chromatograms. The X axis represents the time until elutionfor each component of the sample and the Y axis represents the value ofintensity at 216 nm for each component of the sample, as indicator ofprotein concentration.

FIG. 2. Immuno-detection with an anti EGF antibody conjugated withalkalyne phosphatase, after SDS-PAGE electrophoretic separation for thevaccine composition obtained by ultrafiltration Line 1: Molecular weightmarker, Line 2 vaccine composition.

FIG. 3. Shows the overlapping of the chromatograms corresponding to thehomoligomeric hrEGF-hrEGF conjugated and of the chromatogramcorresponding to a chemical conjugated hrEGF-rP64K without purify. Inthe circle is shown the coincidence of species among both chromatograms.The X axis represents the time for elution of each component and the Yaxis represents the value of intensity at 216 nm of each samplecomponent, as indicator of the protein concentration.

FIG. 4. Chromatographic profile corresponding to the vaccine disclosedby the present invention. The shady area corresponds to the hrEGF-rP64Kconjugated species. The X axis represents the time for elution of eachfraction and the Y axis represents the value of intensity at 216 nm ofeach component in the sample, as indicator of the protein concentration.

FIG. 5. The peptide map of the chromatographic fraction corresponding tothe conjugated hrEGF-rP64K. These are the results from two vaccinecompositions purified independently by an ultrafiltration membrane. TheX axis represents the time and the Y axis represents the units of miliintensity.

FIG. 6. Anti-hrEGF antibody titers in mice immunized with differentimmunogens: Conventional vaccine composition, vaccine compositionpurified by ultrafiltration and permeate proceeding from theultrafiltration purification method. X axis corresponds to the seradilutions from each animal and the Y axis represents the value ofoptical density at 405 nm of each sample, as indicator of the proteinconcentration.

FIG. 7. Chromatographic profile by HPLC-RP obtained during thequantification of the residual glutaraldehyde content of the vaccinecomposition obtained by the purification method using 50 kDaultrafiltration membranes (vaccine composition A) and for the vaccinecomposition using dialysis purification method (vaccine composition B).

1. A vaccine composition comprising the protein conjugated hrEGF-rP64K,wherein the P64K protein molecule is bound to two hrEGF molecules. 2.The vaccine composition according claim 1, wherein the conjugation ratiobetween rP64K carrier protein and the hrEGF is 12.04:24.05 nmolar. 3.The vaccine composition according claim 1, wherein said compositioncomprises a homogenous mix of the conjugated hrEGF-rP64K being free ofmolecular species comprising like hrEGF polymers or free hrEGF.
 4. Thevaccine composition according claim 1, wherein the homogenous mix of theconjugated hrEGF-rP64K is free of glutaraldehyde.
 5. The vaccinecomposition according claim 1, wherein the composition comprises ahomogenous mix of the conjugated hrEGF-rP64K, and further comprises anadjuvant selected from the group comprising of aluminum hydroxide andMontanide.
 6. The vaccine composition according claim 5, wherein theadjuvant comprises Montanide.
 7. The vaccine composition according claim5, wherein the adjuvant comprises aluminum hydroxide.
 8. The vaccinecomposition according claim 1, wherein the composition comprises ahomogenous mix of the conjugated hrEGF-rP64K having the capability ofinducing an increase of at least 10 times the anti-EGF antibodiestitters in the serum of immunized mammal.
 9. The vaccine compositionaccording claim 1, wherein the composition comprises a homogenous mix ofthe conjugated hrEGF-rP64K having the capability of inducing an increaseof at least 10 times the anti-EGF antibodies titters in the serum of ahuman.
 10. The vaccine composition according claim 1, wherein thecomposition comprises a homogenous mix of the conjugated hrEGF-rP64Khaving the capability of inducing an increase of at least 100 times theanti-EGF antibodies titters in the serum of a human.
 11. A procedure forobtaining the vaccine composition of claim 1, under sanitary standardwherein said procedure comprises providing appropriate conditions forthe covalent conjugation of the EGF to the rP64K carrier protein, andpurifying the molecular complex, by ultra filtration, to remove thebiologically inactive conjugated species and other impurities.
 12. Theprocedure of claim 11, wherein the conjugation reaction consists of asingle step of mixing the hrEGF protein previously concentrated (>6mg/mL) and the P64K protein (≧1 mg/mL) in the conjugation reactor, andthen adding to the mixture solution PBS/MgCl2 (pH 6.8-7.2) and theconjugation solution of glutaraldehyde 0.5%, and said mixture isincubated at 22° C.±2° C. with continuous stirring for two hours. 13.The procedure of claim 12, wherein the parameters of conjugation betweenthe proteins hrEGF and rP64K fix the conjugation ratio to two hrEGFmolecules for each rP64K molecule.
 14. The procedure for obtaining thevaccine composition according to claim 11, wherein the conjugatedprotein is purified by a ultra filtration diafiltration method using amembrane having a pore size between 50 and 100 kDa.
 15. The procedurefor obtaining the vaccine composition according to claim 14, wherein theconjugated protein is purified by a ultra filtration diafiltrationmethod using a membrane having a pore size of 50 kDa.
 16. The procedurefor obtaining the vaccine composition according to claim 14, wherein theconjugated protein is purified by a ultra filtration diafiltrationmethod using a membrane having a pore size of 100 kDa.
 17. The vaccinecomposition comprising provided by the procedure of claim
 15. 18. Thevaccine composition provided by the procedure of claim
 16. 19. Thevaccine composition comprising the conjugated protein hrEGFrP64K,wherein the final concentration of the total protein is in the range1-12 mg/mL.
 20. Use of the vaccine composition of claim 17 for themanufacture of a reagent to elicit an immune response against the humanEGF.
 21. Use of the vaccine composition of claim 18 for the manufactureof a reagent to elicit an immune response against the human EGF.
 22. Useof the vaccine composition of claim 17 for the manufacture of a reagentuseful for the treatment of epithelial tumors.
 23. Use of the vaccinecomposition of claim 18 for the manufacture of a reagent useful for thetreatment of epithelial tumors.